In Gratton U.S. Pat. No. 4,840,485, and numerous other references, a method is disclosed of performing cross-correlation frequency domain fluorometry and/or phosphorimetry. This technique is capable of obtaining sophisticated data about phase shifts and modulation changes of luminescence (including phosphorescence) by the use of an amplitude modulated excitation light at a first frequency for the material being tested, coupled with a detector where the gain is modulated at a different frequency. Data is recovered at the cross-correlation frequency, which is the difference between the above two frequencies.
In accordance with this invention, improvements are provided in the simultaneous measurement of the spectral intensity of the luminescence (phosphorescence) of a target material at a number of wavelengths, making use of optical array detector means for example. As is known to the art, an optical array detector can be made using a linear array of detectors or matrix of detectors such as a charge coupled device (CCD) or charge insulator device (CID) chip. For example, to obtain spectral information from the luminescence of a target material excited by a modulated beam of electromagnetic energy, these devices are placed at a focus of an aberration-corrected, flat-field dispersive monochromator or polychromator to create a spectrum. Individual points on the spectrum are then detected at different diodes or other detectors of the array of detectors. As an advantage of this technique, the many detections at various points along the spectrum may be made simultaneously, which provides a significant improvement over a scanning monochromator-based instrument with a single detector, where measurements are sequentially made along the spectrum of luminescence.
One disadvantage of diode array detectors, for example, is that they are generally less sensitive than photomultiplier tubes, since in a diode array a photon can produce only one charge while a photomultiplier tube can have a high electron gain. Also, diode arrays are electronically noisier than photomultipliers, and the dynamic range of diode arrays is less than photomultipliers. Optical array detectors and CCD cameras have been used for more than a decade by several companies in equipment designed for steady-state luminescence measurements (for purposes of this application, the term "luminescence" is intended to include fluorescence). The reading of the device in such analyzers, i.e. The measurement of the intensity of each element of the array, is performed sequentially. Some devices allow random addressing of elements of the array. Thus, it takes a substantial amount of time to read the content of the entire array. The fastest linear array can be read in a fraction of a millisecond, but normally the entire array can be read at a rate of no more than about a hundred times a second. Thus, spectral or spatial variations that occur in a faster time scale than this can not be directly recorded using these devices.
To measure fast spectral changes, for example in the nanosecond range, the above devices may be generally coupled with a fast, gatable, proximity-focused image intensifier. The gain of the intensifier can be changed very rapidly, i.e., in a few nanoseconds, providing a simple method to acquire a time slice or sampling of a repetitive, fast changing signal. The entire time evolution of the process can be measured by varying the delay between the start of the repetitive process and the opening of the gate of the image intensifier. Using this method, the time evolution can be recorded to the shortest time the gate can be opened, which is about five nanoseconds for most of the systems available. Recently, a new technique, based on a RF matched strip on the cathode of the micro-channel plate intensifier has been introduced with a time window of about fifty picoseconds. However, such a sampling method of data collection is very inefficient, since the overall duty cycle, i.e., the time of data collection relative to the total time of a measurement, is very small. For example, to acquire one hundred data points of a time varying process using a time slice of five nanoseconds results in a duty cycle of about 5.times.10.sup.-7 seconds if the array can be read at a maximum speed of 100 Hz. Of course, the duty-cycle problem is not very severe for low repetitive pulsed laser sources, but it becomes of crucial importance when high repetition rate laser sources or sinusoidally intensity modulated sources are employed such as in the K2 Multifrequency Phase Fluorometer (MPF) made by I.S.S..
Recently, microchannel plate detectors (MCP) have been used also in frequency domain fluorometry and/or phosphorimetry. See, for example, Lakowicz, et al. "Gigahertz Frequency Domain Fluorometry: Applications to Picosecond Processes and Future Developments", Time-resolved Laser Spectroscopy in Biochemistry, edited by J. R. Lakowicz, Proceedings S.P.I.E., Vol. 909, p.15-22, (1988); Laczko, et al., "A 10-GHz Frequency-Domain Fluorometer", Rev. Sci. Instrum., Vol 61, pp. 2332-2337, (1990). The MCP-PMT response to short-lived phenomenon is much better since the spread in electron paths in such devices is much less. Typical frequency response ranges to 3 GHz (50% response point) for 6 micron MCP tubes. However, their high internal resistance precludes the use of internal heterodyning although one attempt was made, Berndt, K. W., et al., "4-GHz Internal MCP-Photomultiplier Cross-correlation", Rev. Sci. Instrum., Vol 61, pp. 2557-2565, (1990). One of the drawbacks of non-imaging, photomultiplier tube detectors is their inability to simultaneously process the various regions of a spectrum. One can select various emission wavelengths and obtain spectrally resolved luminescence lifetime information by the above prior art. However, the procedure is quite time consuming since only one emission wave length is acquired at a time.
Micro-channel plate detectors have been used in frequency domain fluorometry and/or phosphorimetry as image intensifiers in Gratton et al., "Parallel Acquisition of Fluorescence Decay Using Array Detectors", Time-resolved Laser Spectroscopy in Biochemistry II, edited by J. R. Lakowicz, Proceedings S.P.I.E., Vol 1204, part 1, p. 21-25 (1990). In this article, the radio frequency gain modulation of a gatable, proximity-focused micro-channel plate image intensifier is optically coupled to a diode array of 512 elements. This system is used with a light source amplitude modulated in the MHz range. Equivalent time-resolution of this instrument is about 100 ps. The frequency response of the instrument was 100 MHz. High frequency information, in the MHz region, is down converted into a low frequency signal of several to tens of Hz by way of internal gain modulation of the detector as discussed in Gratton and Limkeman, "A Continuously Variable Frequency Cross-Correlation Phase Fluorometer with Picosecond Resolution", Biophysical Journal, Vol. 4, p. 315-324 (1983).
This method provides a simple way to conveniently and accurately determine phase and modulation at high frequencies in the MHz range. From the phase and modulation values, the characteristic relaxation times of the system under investigation can be easily obtained using standard methods. A slow readout diode array or charge coupled device detector (with a maximum frame-transfer readout speed of 60 Hz) is combined with the frequency down-conversion capabilities of a fast-gain-modulated proximity-focused image intensifier. Modulation frequencies of the intensity modulated light source and the gain modulated detectors differ by an amount of this cross-correlation or heterodyning frequency. This low frequency signal thus produced by the cross-correlation or heterodyning passes through the phosphor screen, which has a frequency response maximum of about 1 KHz.
The above articles comprise the first description of the use of single-step, internal cross-correlation heterodyning with an array detector in multifrequency phase fluorometry. Such a gain-modulated array detector system overcomes the low duty cycle and some disadvantages of other non-imaging detector systems. As described in Feddersen et al., the system cannot be used other than at selected modulation frequencies and in a relatively narrow range of frequency. The main problem is the strong radio frequency interference of the system and the low integration capability.
By this invention, an apparatus and method for cross-correlation frequency domain fluorometry-phosphorimetry is provided which exhibits significant improvements in the speed of data acquisition at multiple points across a luminescence spectrum. Thus, the apparatus of this invention can be used to derive data from a decaying spectrum under non-steady state conditions in a manner showing significant improvements over detectors of the prior art. Time resolutions in the picosecond time range may be achieved when modulation frequencies in accordance with this invention are in the 100 to 200 MHz range, so that transient, unstable luminescence phenomena may be studied. Also, the problems arising from radio-frequency interference present in the apparatus described by Gratton et al. are here completely resolved by use of a novel double modulation scheme.